Six Types of Enzyme Catalysts

Although a huge number of reactions occur in living systems, these reactions fall into

only half a dozen types. The reactions are:

1. Oxidation and reduction. Enzymes that carry out these reactions are called

oxidoreductases. For example, alcohol dehydrogenase converts primary

alcohols to aldehydes.

In this reaction, ethanol is converted to acetaldehyde, and the cofactor, NAD, is

converted to NADH. In other words, ethanol is oxidized, and NAD is reduced.

(The charges don't balance, because NAD has some other charged groups.)

Remember that in redox reactions, one substrate is oxidized and one is

reduced.

2. Group transfer reactions. These enzymes, called transferases, move

functional groups from one molecule to another. For example, alanine

aminotransferase shuffles the alpha-amino group between alanine and

aspartate:

3. Other transferases move phosphate groups between ATP and other compounds,

sugar residues to form disaccharides, and so on.

4. Hydrolysis. These enzymes, termed hydrolases, break single bonds by adding

the elements of water. For example, phosphatases break the oxygen-

phosphorus bond of phosphate esters:

5. Other hydrolases function as digestive enzymes, for example, by breaking the

peptide bonds in proteins.

6. Formation or removal of a double bond with group transfer. The functional

groups transferred by these lyase enzymes include amino groups, water, and

ammonia. For example, decarboxylases remove CO2 from alpha- or beta-keto

acids:

Dehydratases remove water, as in fumarase (fumarate hydratase):

Deaminases remove ammonia, for example, in the removal of amino groups

from amino acids:

7. Isomerization of functional groups. In many biochemical reactions, the

position of a functional group is changed within a molecule, but the molecule

itself contains the same number and kind of atoms that it did in the beginning.

In other words, the substrate and product of the reaction are isomers. The

isomerases (for example, triose phosphate isomerase, shown following), carry

out these rearrangements.

8. Single bond formation by eliminating the elements of water. Hydrolases

break bonds by adding the elements of water; ligases carry out the converse

reaction, removing the elements of water from two functional groups to form a

single bond. Synthetases are a subclass of ligases that use the hydrolysis of ATP

to drive this formation. For example, aminoacyl-transfer RNA synthetases join

amino acids to their respective transfer RNAs in preparation for protein

synthesis; the action of glycyl-tRNA synthetase is illustrated in this figure:

The Michaelis-Menten equation

If an enzyme is added to a solution containing substrate, the substrate is converted to

product, rapidly at first, and then more slowly, as the concentration of substrate

decreases and the concentration of product increases. Plots of substrate (S) or product

(P) against time, called progress curves, have the forms shown in Figure

1 . Note that the two progress curves are simply inverses of each other. At the end of

the reaction, equilibrium is reached, no net conversion of substrate to product occurs,

and either curve approaches the horizontal.

Figure 1

Another way to look at enzymes is with an initial velocity plot. The rate of reaction is

determined early in the progress curve—very little product is present, but the enzyme

has gone through a limited number of catalytic cycles. In other words, the enzyme is

going through the sequence of product binding, chemical catalysis, and product release

continually. This condition is called the steady state. For example, the three curves in

Figure

2 represent progress curves for an enzyme under three different reaction conditions. In

all three curves, the amount of enzyme is the same; however, the concentration of

substrate is least in curve (a), greater in curve (b), and greatest in curve (c). The

progress curves show that more product forms as more substrate is added. The slopes

of the progress curves at early time, that is, the rate of product formation with time

also increase with increasing substrate concentration. These slopes, called the initial

rates or initial velocities, of the reaction also increase as more substrate is present

so that:

The more substrate is present, the greater the initial velocity, because enzymes act to

bind to their substrates. Just as any other chemical reaction can be favored by

increasing the concentration of a reactant, the formation of an enzyme-substrate

complex can be favored by a higher concentration of substrate.

Figure 2

A plot of the initial velocities versus substrate concentration is a hyperbola (Figure

3 ). Why does the curve in Figure 3 flatten out? Because if the substrate concentration

gets high enough, the enzyme spends all its time carrying out catalysis and no time

waiting to bind substrate. In other words, the amount of substrate is high enough so

that the enzyme is saturated, and the reaction rate has reached maximal velocity,

or Vmax. Note that the condition of maximal velocity in Figure 3 is not the same as the

state of thermodynamic equilibuium in Figures 1 and 2 .

Figure 3

Although it is a velocity curve and not a binding curve, Figure

3 is a hyperbola. Just as myoglobin is saturated with oxygen at high enough pO2, so an

enzyme is saturated with substrate at high enough substrate concentration, designated

[S]. The equation describing the plot in Figure 2 is similar in form to the equation used

for O2 binding to myoglobin:

Km is the Michaelis constant for the enzyme binding substrate. The Michaelis constant

is analogous to, but not identical to, the binding constant for the substrate to the

enzyme. Vmax is the maximal velocity available from the amount of enzyme in the

reaction mixture. If you add more enzyme to a given amount of substrate, the velocity

of the reaction (measured in moles of substrate converted per time) increases, because

the increased amount of enzyme uses more substrate. This is accounted for by the

realization that Vmax depends on the total amount of enzyme in the reaction mixture:

where Et is the total concentration of the enzyme and kcat is the rate constant for the

slowest step in the reaction.

Other concepts follow from the Michaelis-Menten equation. When the velocity of an

enzymatic reaction is one-half the maximal velocity:

then:

because:

In other words, the Km is numerically equal to the amount of substrate required so that

the velocity of the reaction is half of the maximal velocity.

Alternatively, when the concentration of substrate in the reaction is very high (Vmax

conditions), then [S] >> Km, and the Km term in the denominator can be ignored in the

equation, giving:

On the other hand, when [S]

In the terms of the Michaelis-Menten equation, inhibitors can raise Km, lower Vmax, or

both. Inhibitors form the basis of many drugs used in medicine. For example, therapy

for high blood pressure often includes an inhibitor of the angiotensin converting

enzyme, or ACE. This enzyme cleaves (hydrolyzes) angiotensin I to make angiotensin

II. Angiotensin II raises blood pressure, so ACE inhibitors are used to treat high blood

pressure. Another case is acetylsalicylic acid, or aspirin. Aspirin successfully treats

inflammation because it covalently modifies, and therefore inactivates, a protein

needed to make the signaling molecule that causes inflammation.

The principles behind enzyme inhibition are illustrated in the following examples.

Alkaline phosphatase catalyzes a simple hydrolysis reaction:

Phosphate ion, a product of the reaction, also inhibits it by binding to the same

phosphate site used for binding substrate. When phosphate is bound, the enzyme

cannot bind substrate, so it is inhibited by the phosphate. How to overcome the

inhibitor? Add more substrate: R –O –PO32-. Because the substrate and the inhibitor

bind to the same site on the enzyme, the more substrate that binds, the less inhibitor

binds. When is the most substrate bound to the enzyme? Under Vmax conditions.

Phosphate ion reduces the velocity of the alkaline phosphate reaction without reducing

Vmax. If velocity decreases, but Vmax doesn't, the only other thing that can change is Km.

Remember that Km is the concentration where v= Vmax/2. Because more substrate is

required to achieve Vmax, Km must necessarily increase. This type of inhibition, where

Km increases but Vmax is unchanged, is called competitive because the inhibitor and

substrate compete for the same site on the enzyme (the active site).

Other cases of inhibition involve the binding of the inhibitor to a site other than the site

where substrate binds. For example, the inhibitor can bind to the enzyme on the

outside of the protein and thereby alter the tertiary structure of the enzyme so that its

substrate binding site is unable to function. Because some of the enzyme is made

nonfunctional, adding more substrate can't reverse the inhibition. Vmax, the kinetic

parameter that includes the Et term, is reduced. The binding of the inhibitor can also

affect Km if the enzyme-inhibitor complex is partially active. Inhibitors that alter both

Vmax and Km are called noncompetitive; the rare inhibitors that alter Vmax only are

termed uncompetitive.

You can visualize the effects of inhibitors using reciprocal plots. If the Michaelis-Menten

equation is inverted:

This equation is linear and has the same form as:

so that a plot of 1/ v versus 1/[S] (a Lineweaver-Burk plot, shown in Figure

4 ) has a slope equal to Km/Vmax and a y-intercept equal to 1/Vmax. The x-intercept of a

Lineweaver-Burk plot is equal to-1/Km.

Figure 4

Competitive inhibitors decrease the velocity of an enzymatic reaction by increasing

the amount of substrate required to saturate the enzyme; therefore, they increase the

apparent Km but do not affect Vmax. A Lineweaver-Burk plot of a competitively inhibited

enzyme reaction has an increased slope, but its intercept is unchanged.

Noncompetitive inhibitors both increase the apparent Km and reduce the apparent

Vmax of an enzyme-catalyzed reaction. Therefore, they affect both the slope and the y-

intercept of a Lineweaver-Burk plot, as Figures

5 and 6 show. Uncompetitive inhibitors, because they reduce Vmax only, increase the

reciprocal of Vmax. The lines of the reciprocal plot are parallel in this case.

Figure 5

Figure 6

Covalent inhibition involves the chemical modification of the enzyme so that it is no

longer active. For example, the compound diisopropylfluorophosphate reacts with many

enzymes by adding a phosphate group to an essential serine hydroxyl group in the

enzymes' active sites. When phosphorylated, the enzyme is totally inactive. Many

useful pharmaceutical compounds work by covalent modification. Aspirin is a covalent

modifier of enzymes involved in the inflammatory response. Penicillin covalently

modifies enzymes required for bacterial cell-wall synthesis, rendering them inactive.

Because the cell wall is not able to protect the bacterial cell, the organism bursts easily

and is killed.